Independent Recruitment of a Flavin-Dependent Monooxygenase for Safe Accumulation of Sequestered Pyrrolizidine Alkaloids in Grasshoppers and Moths

Abstract

Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

Molecular characterization, expression pattern and metabolic activity of flavin‐dependent monooxygenases inSpodoptera exigua

“…Three FMO genes were identified from S. exigua , the same number as in B. mori and H. armigera , according to the prediction using genomic data (Langel & Ober, ; Wang et al ., ). Phylogenetic analysis showed that there was no orthologous relationship between insect and mammalian FMOs (Naumann et al ., ), and the branching pattern of the tree displayed two obvious clusters of insect FMOs far separated from human FMOs.…”

“…However, the xenobiotic metabolism by insect FMOs is less studied, with only a few reports from Dr Ober's laboratory. Their research was mainly focused on the detoxification of host plant‐acquired PAs in insects (Naumann et al ., ; Sehlmeyer et al ., ; Langel & Ober, ; Wang et al ., ). PAs are typical plant secondary metabolites thought to play an important role as defensive chemicals in plants and in their adaptation to insect herbivores.…”

“…The enzyme encoded by TjFMO was found to have high N‐oxidation activity on pyrrolizidine alkaloids (PAs), and to transform toxic PAs from host plants to their nontoxic N‐oxides leading to relatively safe accumulation (Langel & Ober, ). Since then, FMOs from moths and grasshoppers have been characterized as a result of research on PA detoxification (Sehlmeyer et al ., ; Wang et al ., ). In the present study we identified three FMO genes from a vegetable insect pest, S. exigua , and analysed their expression characteristics and metabolic activities on insecticides.…”

Climate and host‐plant associations shaped the evolution of ceutorhynch weevils throughout the Cenozoic

“…2012; Wang et al. 2012). However, with our current dataset, we cannot yet ascertain if the proposed coevolutionary patterns observed in ceutorhynch weevils either simply support a “sequential evolution” model, where a single colonization of a new host plant group promoted diversification of the colonizing insect taxa, or if the inferred insect‐host associations may constitute a reciprocal system of coevolutionary radiations, as predicted by the “escape‐and‐radiate” model.…”

Expression plasticity and evolutionary changes extensively shape the sugar‐mimic alkaloid adaptation of nondigestive glucosidase in lepidopteran mulberry‐specialist insects

“…Previous studies revealed that the adaptive plasticity of metabolic enzymes involved in detoxication and digestion is a universal mechanism that is employed by herbivorous insects (Crossley, Chen, Groves, & Schoville, ; Despres, David, & Gallet, ; Heidel‐Fischer & Vogel, ). For the alkaloid resistance, enzymes, including ATP‐binding cassette (ABC) transporters, flavin‐dependent monooxygenase and sucrase showed evolutionary plasticity for alkaloid resistance (Wang, Beuerle, Timbilla, & Ober, ; Wang et al., ; Yu et al., ). Apart from the enzymes for detoxication and digestion, enzymes with other important physiological functions are also revealed here to undergo adaptive evolution to minimize the inhibitory effects.…”